Myosin light chain kinase (MLCK) catalyzes the Ca2+/calmodulin-dependent phosphorylation of the regulatory light chain (RLC) of the motor protein, myosin II. There are two genes for MLCKs. One expresses smooth muscle MLCKs in all cells, including skeletal muscle fibers, whereas the other expresses a distinct skeletal muscle MLCK only in adult skeletal muscle fibers. Smooth muscle MLCK phosphorylates smooth and nonmuscle RLC but not skeletal muscle RLC. However, skeletal muscle MLCK readily phosphorylates all RLCs. In skeletal muscle Ca2+ activation of contraction is mediated through a thin filament regulatory system, troponin-tropomyosin. Recent reports indicate that Ca2+/calmodulin formation is low relative to the total cellular calmodulin and may be limiting for activation of multiple target proteins. Experiments in intact muscles show a correlation between RLC phosphorylation and post-tetanic potentiation of isometric force amplitude or treppe in fast-twitch skeletal muscles. The relative importance of RLC phosphorylation-force relationship is unclear due to modest increases in the rate and extent of force development in skinned fibers and to other factors affecting contractile performance in intact muscles including length-dependent effects on Ca2+ release from the sarcoplasmic reticulum, inter-myofilament spacing and calmodulin regulation of Ryr1, the calcium release channel. The research described in this application will determine (1) the relationship between the free Ca2+ concentration and Ca2+/ calmodulin formation in C2C12 myotubes and enzymatically dispersed mouse skeletal muscle fibers with biosensor molecules, (2) the temporal and spatial distributions of Ca2+/calmodulin binding to and activation of unique biosensor skeletal and smooth muscle MLCKs in skeletal muscle fibers from transgenic animals, and (3) if RLC phosphorylation and increased force amplitude are inhibited in fast-twitch skeletal muscle fibers from mice with ablation of the skeletal muscle MLCK gene. The results from these investigations will provide insights into Ca2+-dependent mechanisms recruited during exercise that enhance muscle performance.